1) Field of the Invention
This invention relates to water-soluble extracts from a plant of Solanum genus, the preparation processes thereof, and pharmaceutical compositions comprising the same.
2) Description of the Related Art
Cancer is one of the major causes of human death globally, and lung cancer, liver cancer, and breast cancer are most common. Although the mechanism of cancer development has yet to be fully understood, it is believed that the onset of cancer in a subject may be caused by abnormal and uncontrollable cell division occurring in said subject (Chen, P. L., et al. (1990), Science, 250, 1576–1580; Finlay, C. A., et al. (1989), Cell, 57, 1083–1093, and Baker, S. J., et al. (1990) Science, 249, 912–915).
Usually, the growth and differentiation of cells in a human or animal body are strictly controlled by growth hormones present in the human or animal body. When cells accumulate therein gene mutations caused by intrinsic and/or extrinsic factors, said cells will generate incorrect signal transmissions, which in turn lead to the uncontrollable growth and division of cells, thereby resulting in the formation of cancer cells gradually (Kerr, J. F. R. (1971) J. Pathol., 105, 13–20).
In recent years, investigators around the world have endeavored to research works of cancers. However, the currently developed and employed cancer therapies fail to provide satisfactory therapeutic effects. In addition to patients' personal factors, the serious side effects of anti-cancer drugs and the resistance of cancer cells to such drugs are the primary problems encountered in clinical therapy.
In view of the fact that the known western medicines employed in clinic fail to effectively improve the current therapies for cancer dieases, some researchers have, based on the investigation results of the onset mechanism of cancer disease, attempted to find active ingredients from traditional Chinese medicines (TCM) or herbs that can be used to cure or relieve the symptoms of cancers.
Apoptosis is considered to be a natural mechanism that regulates animal cell growth (Martin, S. J. and Green, D. R. (1995), Crit. Rev. Oncol. Hemat., 18, 137–153), and it plays an important role in regulating natural cell death, such as the natural tissue shrinkage and absorption occurring during the growth process of animals. In addition, when human cells are damaged and cannot be repaired, apoptosis will be initiated so as to avoid the formation of cancer cells.
The major morphological features of apoptosis include: formation of apoptotic bodies, chromatin condensation, and DNA fragmentation (Arends, M. J. and Wyllie, A. H. (1991) Int. Rev. Exp. Pathol., 32, 223–254; Dive, C., et al., (1992) Biochim. Biophys. Acta 1133, 275–285; and Darzynkiewicz, Z., et al. (1992) Cytometry, 13, 795–808). During apoptosis, debris of dead cells will be rapidly ingested by neighboring cells and macrophages via phagocytosis without inducing an inflammatory response (Sarraf, F. E. and Bowen, I. D. (1988) Cell Tissue Res. 21, 45–49). In addition, when the variation of cell cycle is detected by flow cytometry, the presence of a sub-G1 peak can be observed (Alzerreca, A. and Hart, G. (1982) Toxicology Lett. 12, 151–155; and Lin, C. N., et al. (1986) J. Taiwan Pharm. Assoc. 38, 166). Thus, the sub-G1 peak is considered to be a typical marker for identifying cells that are undergoing apoptosis.
It is reported in literature that cells will become cancer cells if the apoptotic mechanism thereof is out of control (Carson, D. A. and Ribeiro, J. M. (1993) Lancet 341, 1251–1254; and Kaufmann, S. H. (1989) Cancer Res. 49, 5870–5878). Therefore, apoptosis has become a subject of study in oncology. In addition, it is reported that apoptosis can be induced by certain anti-cancer drugs (Wyllie, A. H., et al., (1980) Int. Rev. Cytol. 68, 251–306; Wyllie, A. H., et al., (1984) J Pathol. 142, 67–77; Barry, M. A., et al. (1990) Biochem. Pharmacol., 40, 2353–2362; and Hickman, J. A. (1992) Cancer Metast. Rev., 11, 121–139). Thus, apoptosis points to a major direction in the global development of anti-cancer drugs.
Use of traditional Chinese medicines or herbal medicines to treat diseases has a long history. At present, not a few researchers are trying to find useful anti-cancer drugs from traditional Chinese medicines or herbal medicines. However, the application of traditional Chinese medicines or herbal medicines is still based on empiricism, and is not supported by sufficient scientific evidence. In addition, because the extraction of active ingredients, and the dosage and quality control of traditional Chinese medicines or herbal medicines are not scientized, the therapeutic effects exhibited by the medicines are not consistent.
Furthermore, most of the active ingredients from traditional Chinese medicines or herbal medicines are water insoluble. When water-insoluble material is orally administered to or injected into animal bodies, the intended therapeutic effect thereof may not be achieved due to difficulty in absorption. These are the major restraints that hamper the development and application of traditional Chinese medicines and herbal medicines.
Plants that can be used as medicines are numerous. It is well known that many protein inhibitors extracted from plant materials are used in anti-cancer therapy. Among these protein inhibitors with anti-cancer potential, steroidal alkaloids from a plant of Solanum genus are found to be a potential anti-cancer drug.
It is known that Solanum incanum L. (also known as Solanum incanum Ruiz. & Pav., Solanum coagulans Forsskal in Latin, and bitter apple in English) contains steroidal glycoalkaloid (Kuo, K. W., et al. (2000), Biochemical Pharmacology, 60 (12): 1865–73). In addition, many plants of the Solanum genus are reported to contain steroidal glycoalkaloid, including, for example, Solanum indicum, Solanum nigrum, also known as Long Kui in Chinese and black nightshade in English (Hu, K., et al. (1999), Planta Medica, 65 (1): 35–8), Solanum capsicastrum (known as false Jerusalem cherry in English), Solanum xanthocarpum, Solanum melongena (Blankemeyer, J. T., et al. (1998), Food &Chemical Toxicology, 36 (5): 383–9), Solanum coagulans, Solanum tuberosum (Friedman, M., et al. (1996), Journal of Nutrition, 126 (4): 989–99), Solanum sodomeum (known in Australia as apple of Sodom), Solanum turburosum, Solanum aculeastrum (Wanyonyi, A. W., et al. (2002), Phytochemistry, 59 (1): 79–84), Solanum lycocarpum (Peters, V. M., et al. (2001), Contraception, 63 (1):53–5), Solanum khasianum (Putalun, W., et al. (2000), Biological &Pharmaceutical Bulletin, 23 (1): 72–5), Solanum suaveolens (Ripperger, H., et al. (1997), Phytochemistry, 46 (7): 1279–82), Solanum uporo (Ripperger, H., et al. (1997), Phytochemistry, 44, (4): 731–4), Solanum abutiloides (Tian, R. H., et al. (1997), Phytochemistry, 44 (4): 723–6), Solanum coccineum (Lorey, S., et al. (1996), Phytochemistry, 41 (6): 1633–5), Solanum unguiculatum (Sarg, T. M., et al. (1995), Pharmacy World &Science, 17 (6): 191–4), Solanum robustum (Ripperger, H. (1995), Phytochemistry 39 (6): 1475–7), Solanum anguivi (Ripperger, H., et al. (1994), Phytochemistry, 37 (6): 1725–7), Solanum platanifolium (Puri, R., et al. (1994), Journal of Natural Products 57 (5): 587–96), Solanum mammosum (Alzerreca, A., et al. (1982), Toxicology Letters, 12 (2–3): 151–5), etc.
Up to the present, steroidal alkaloids which can be obtained from the aforesaid plants of Solanum genus comprise, for example, solamargine, solasonine, khasianine and solasodine (Chataing, B., et al. (1998), Planta Medica 64, 31–36, and Weissenberg, M., et al. (1998), Phytochemistry 47, 203–209). The structures of solasonine and solamargine are as follows:

In addition, studies have shown that solamargine obtained from various plant materials is capable of inhibiting growth of the following organisms: parasites, such as Trypanosoma cruzi; insects, such as Tribolium castaneum (known as red flour beetle), Manduca sexta (known as tobacco hornworm); mold, such as Phoma medicaginis and Rhizoctomia solani; and mollusks, such as Lymnaea cubensis and Biomphalaria glabrata (Chataing, B., et al. (1998), Planta Medica, 64, 31–36; Fewell, A. M., et al. (1994), Phytochemistry, 37, 1007–1011; Lin, C. N., et al. (1990), J. Nat. Prod., 53, 513–516).
Furthermore, Chun-Nan Lin et al. reported that solamargine can be obtained from the fruit of Solanum incanum, and the structure thereof belongs to steroidal alkaloid glycoside. It is found that the compound protects the liver from CCl4-induced damage, and inhibits the growth of JTC-26 and human PLC/PRF/5 hepatoma cells (Lin, C. N. et al. (1986), J. Natural Prod., 53, 513–516).
Shu-Hui Hsu et al. studied the mechanism of cytotoxicity of solamargine, and found that solamargine increases death of cells, such as Hep3B and normal skin fibroblast cells, by apoptosis pathway. Particularly, it was found that the gene expression of TNF receptor I involved in the process of cell apoptosis was up-regulated by solamargine (Hsu, S. H. et al. (1996), Biochem. Biophys. Res. Comm., 229, 1–5).
Katsuya Fukuhara and Isao Kubohas reported in Phytochemistry, 30 (2): 685–687, 1991, that ripe fruits of Solanum incanum were extracted with methanol at room temperature. Then, the solvent was removed under reduced pressure, and the residue was lyophilized to give a dark brown extract. Next, the extract was suspended in water containing methanol (1%). After removing the water insoluble portion, the suspension was partitioned with n-hexane, chloroform, ethyl acetate, and water, and an aqueous layer with bioactivity was obtained. The aqueous layer with bioactivity thus obtained was subsequently subjected to rotation locular countercurrent chromatography and droplet countercurrent chromatography such that solamargine and solasonine, the two major compounds, were obtained.
It was disclosed by Ke Hu et al. (1999) in Planta Medica 65, 35–38, that a dried whole herb of Solanum nigrum was refluxed with 75% EtOH. The solvent was removed in vacuo to obtain a brown residue, which was defatted with petroleum ether to give an extract. The resultant extract was suspended in water and was subjected to chromatography on a macroresin column. There is an active compound present in 60% EtOH eluent. 60% EtOH eluent was then partitioned with H2O and extracted with n-BuOH, and the n-BuOH extract thus obtained was subjected to column chromatography on silica gel using CHCl3—MeOH—H2O as an eluant and on Sephadex LH-20 using MeOH—H2O (60:40) as an eluant, yielding β2-solamargine, solamargine and degalactotigonin. However, this paper did not teach how a water-soluble bioactive extract can be obtained from Solanum nigrum. 
EP 0 020 029 A1 disclosed that a plant material of Solanum sodomeum, known in Australia as apple of Sodom, was extracted with a diluted acid solution, such as 2% or 3% acetic acid, to obtain a first acidic extract (supernatant portion), and the solid residue was then extracted with another volume of the diluted acid solution after being separated from the first acidic extract so as to yield a second acidic extract (supernatant portion). After combining the first and second acidic extracts, a base was added to obtain a precipitate. The precipitate was dissolved in boiling ethanol. After removal of ethanol, a fine powder extract (referred to as BEC 001) was obtained. BEC 001 extract was further separated and purified to yield various glycoalkaloids, including solamargine, solasonine, and mono- and di-glycosides of soladodine.
Although EP 0 020 029 A1 mentioned that H2O can be used as a carrier for BEC 001 extract, the extract was essentially formulated with dimethyl sulfoxide solution (DMSO), paraffin, zinc ointment, zinc cream, and cetomacrogol (a surfactant) in the working examples of said patent.
Furthermore, according to U.S. Pat. No. 5,958,770, solasodine glycosides used in the cytotoxic experiment in vitro were first dissolved in DMSO and then diluted to give a 5% DMSO solution. In addition, solasodine glycosides employed in the experiment were either in a form of a mixture (referred to as BEC) including solamargine (33%), solasonine (33%), and di- and mono-glycoside (34%), or in a form of a separate component (solamargine, solasonine, a mixture of di- and mono-glycoside, and the aglycones of solasodine).
Since the aforesaid steroidal alkaloids are water-insoluble, alcohol distillation is a common extracting method used in the aforesaid patents or literatures, and the extracted portions are usually dissolved in an organic solvent, i.e. DMSO for analysis. Because water-insoluble materials are not suitable for direct injection into animal bodies and may not be absorbable by the digestive tract during oral administration, the therapeutic effects of steroidal alkaloids cannot be achieved, thereby limiting the pharmaceutical application and development of steroidal alkaloids.
The applicant found that the dried powders of solamargine and/or solasonine definitely could not dissolve in water without being pre-treated with DMSO, and may not completely dissolve in water even after being treated with DMSO. Specifically, steroidal alkaloids extracted by using the method disclosed in EP 0 020 029 A1 could not dissolve in distilled water. Although solamargine or solasonine can dissolve in water after being first dissolved in DMSO, they will be precipitated if the concentration thereof is too high (more than 5 mg/ml). In addition, DMSO (>1%) per se has a strong cytotoxicity to cells, and thus, the concentration thereof should be controlled to be less than 5%. Such facts clearly indicate that the use of DMSO organic solvent to dissolve steroidal alkaloids extracted from a plant of Solanum genus has limitations.
In view of the foregoing, at present, steroidal alkaloids are primarily produced by chemical manufacturers on a limited and small scale basis and in single batches, and there is not an efficient process for extracting water-soluble steroidal alkaloids from a plant of Solanum genus on a large scale for commercial use. As such, the application of steroidal alkaloids in the manufacture of medicaments and drugs is limited.